Traditional Sequencing By Synthesis technologies typically involve separate incorporation, washing, imaging, and cleavage steps with continuous feeding of multiple buffers. 454 Bio’s One Pot sequencing on the LED-TIRF based Transformer only needs one solution without any fluid exchange for imaging, washing, or cleaving steps.
One Pot Sequencing on the LED-TIRF Transformer takes place at a constant temperature of 58 C. A cycle of sequencing consists of a 10 minute incubation to allow for Lightning Terminator incorporation. Next, the 4 visible wavelength LEDs are pulsed to excite each Lightning Terminator. Images are captured in real time. After images are taken to record which base was incorporated, the Lightning Terminators™ are photocleaved by the UV LED. The linked dyes on incorporated Lightning Terminators™ are cleaved and diffuse away, allowing for the next base incorporation. This process is repeated for each cycle of sequencing.
This protocol describes the procedure to set up One Pot Sequencing on the LED-TIRF Transformer.
|Description of Change
|One pot Sequencing
Materials and Equipment
|One pot buffer
|Lightning Terminator™ mix
|Sequencing Primer: ACACTCTTTCCCTACACGACGCTCTTCCGATC*T
|Standard desalted lyophilized DNA oligo; 100 nmole minimum scale; * indicates phosphorothioated DNA bases
|Pipettes and tips (1000 µL, 200 µL, 20 µL, 2 µL)
Notes before starting
Lightning Terminators™ need to be carefully handled and only exposed to shorter than 500 nm light. Care should be taken to protect Lightning Terminators™ from photobleaching and from photocleaving due to exposure to light.
- Open the sequencing setup worksheet.
- Enter the required information based on your experiment plan.
- Retrieve OPB buffer, Seq Primer, LTMix, DISCS from -20°C freezer and thaw them by leaving them on the bench for 30 minutes at room temperature.
- Pre-heat Transformer to 65°C.
- Seq Primer Hybridization:
- Prepare sequencing primer hybridization solution following the worksheet.
- Add 120 µL hybridization solution into the sequencing reservoir.
- Load the reservoir onto the pre-heated 454 Bio Transformer.
- Incubate reservoir at 65°C for 10 minutes.
- Bring the reservoir to the bench and incubate at room temperature for 10 minutes
- Set Transformer temperature to 58°C for sequencing.
- Rinse reservoir with 120 µL OPB once.
- Therminator Preloading:
- This step can be skipped if circular DISCS or RNA DISCS is used.
- Prepare Therminator solution following the worksheet.
- Add 60 µL Therminator solution into the reservoir.
- Incubate for 3 minutes at room temperature.
- Rinse the reservoir with 120 µL OPB once.
- Ensure Transformer temperature has reached 58° C
- Prepare sequencing solution using the worksheet.
- Mix the solution thoroughly by vortexing and spinning. Tap the tube with fingers to remove any bubbles.
- Carefully add the solution to reservoir avoiding any bubbles.
- Load the reservoir on the Transformer.
- Set a timer for 10 minutes for the first cycle incorporation.
- After 10 minutes, manually adjust the focus for each wavelength:
- In the manual controls, select a filter and pulse time and then start the preview.
- Use the focus adjustment buttons to bring the clusters in focus.
- Stop the preview.
- Verify that all other wavelengths are in focus by repeating steps 13.1 and 13.3 for each wavelength. It should not be necessary to refocus any wavelength.
- If any of the wavelengths are out of focus, calibrate your instrument’s focus before continuing.
- Repeat steps 13.1 to 13.3 for each wavelength After four wavelengths’ cluster images are all in focus, switch the focus pin to auto position.
- Start the protocol and one pot sequencing will automatically finish all desired cycles.